Methyl parathion hydrolase (MPH) can degrade a wide range of organophosphorus compounds, but its efficiency in hydrolysing chlorpyrifos, one of the most popular pesticides applied for crop protection, is much lower than that in hydrolysing the preferred substrate methyl parathion. In this study, random mutagenesis was adopted to improve MPH to enhance its efficiency in hydrolysing the poorly hydrolysed substrate chlorpyrifos. Rapid screening of the improved MPH variants was carried out using Bacillus subtilis WB800 secretory expression system to investigate the distribution of improved MPH variants based on the size of clear haloes as a result of chlorpyrifos hydrolysis. Four improved MPH variants were isolated, and one variant K3, in particular, showed a 5-fold increase in kcat value for chlorpyrifos hydrolysis. Furthermore, most of the MPH variants obtained in this study possessed enhanced thermostability and pH stability. The approaches adopted in this study could be extended to create other MPH variants with increased activity for hydrolysing other poorly hydrolysed substrates.
Significance and Impact of the Study
Chlorpyrifos is one of the toxic organophosphorus compounds (OP compounds) widely used for insecticides control. Water, soil and foodstuff have been contaminated seriously by chlorpyrifos in some areas. It is urgent to find effective methods to remove its contamination. This work contributes to improve methyl parathion hydrolase (MPH) to enhance its efficiency in hydrolysing the poorly hydrolysed substrate chlorpyrifos. Our study brings new insights for enzymatic strategy for the decontamination of toxic OP compounds.